PROJECT 1 ABSTRACT Ionizing radiation (IR) is used extensively to treat lung cancer, but IR can damage healthy lung tissue, causing radiation-induced lung injury (RILI). When unmitigated, RILI leads to radiation-induced lung fibrosis (RILF). RILF causes deterioration of pulmonary function and respiratory failure, and upwards of 30% of patients develop RILF as a complication to radiotherapy. Investigation into treatments to block or mitigate the manifestation of RILF is of significant importance to limit chronic lung injury following radiotherapy. Despite progress in recent years, countermeasures to effectively combat or prevent RILF do not exist in the clinic, which represents a significant deficiency to treat the long-term effects of IR. This deficiency is becoming even more significant because immunotherapy, which is rapidly being incorporated into many standard chemo- and radiotherapy regimens, may also induce and exacerbate pulmonary fibrosis. In response to radiation-induced cellular injuries, a proinflammatory environment is observed in damaged tissues. In particular, elevated levels of tumor necrosis factor alpha (TNF-?) and transforming growth factor beta (TGF-?) play key roles in chronic oxidative damage and fibrogenesis following lung exposure to IR. Indeed, studies hindering TNF-? or TGF-? pathways ameliorate oxidative damage, inflammation, and pulmonary fibrosis, while the actions of both of these cytokines lead to lung fibrosis. Because of the cytokine-mediated cellular damage of TNF-? and the profibrotic effects of TGF-?, both cytokines play a role in the development of RILF. We hypothesize that simultaneously impairing TNF-? and TGF- ? pathways will more effectively combat IR-induced damage to healthy lung tissue and provide better protection against RILF than impairing either cytokine separately. In our laboratory, we recently developed a small-molecule anticytokine?Tk-1?that inhibits the TGF-? receptor 1 (TGF?R1) and impairs TNF-? production by inhibiting mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) (IC50 = 0.001 M [TGF?R1] and 0.005 M [MAP4K4]). We hypothesize that this novel anticytokine, acting synergistically to impair IR-induced cytokine induction, will diminish proinflammatory TNF-? signaling in irradiated tissue and ameliorate TGF-??mediated fibrosis. We will test this hypothesis in cell and animal-based models of RILF. Completion of this proposal will help generate the data necessary for R01-level funding and may uncover a novel clinical strategy to mitigate IR- induced pulmonary fibrosis.